The overall objective of this project is to investigate the active site of monoamine oxidase (MAO) and to unravel the molecular basis for the catalytic differences between the two types of MAO (MAO-A and B). This investigation will be conducted with the two photoaffinity labeling probes developed in this laboratory: 4-fluoro-3-nitrophenyl azide (FNPA) and 2 nitro-4-azidophenyl-dopamine (NAP-dopamine). FNPA has been shown to be a selective photoaffinity labeling probe for MAO-B, whereas NAP-dopamine was found to be able to inhibit both MAO-A and B photodependently. In addition, we have found that FNPA labels the active site of MAO-B as regions different from the FAD binding site, namely, the substrate binding site. In this application, the [H3]FNPA label d active site peptides produced by complete tryptic-chymotryptic digestion of the [H3]FNPA photolabeled beef liver MAO-B will be purified by reverse phase HPLC. The amino acid sequence of these labeled peptides will be determined by microsequencing techniques. This study will provide the first information on the substrate binding site of MAO-B. Furthermore, purified human placenta MAO-A and purified beef liver MAO-B will be labeled by [H3]NAP-dopamine. The biochemical nature of NAP-dopamine labeling site(s) on two MAO's will be characterized systematically by a series of protection experiments utilizing specific substrates and inhibitors of MAO. [H3]NAP-dopamine labeled active site peptides will be isolated by reverse phase HPLC and the amino acid sequences will be determined. This study will provide unequivocal evidence to demonstrate the structure differences that exist between MAO-A and -B. This investigation also has significant clinical application since MAO is an important enzyme in the metabolism of monoamine neurotransmitters. The level of MAO is altered in patients with certain mental disorders. Better understanding of the chemical nature of this enzyme and the molecular basis of the two types of MAO will help to better understand the mental diseases associated with this enzyme.